We attempted to define the immune response to the dengue type 4, strain 814669, envelope glycoprotein (E) in Balb/c (H-2(d)) and CBA/N or Balb/K (H-2(k)) mice using 38 synthetic 15-amino-acid peptides that span the E sequence (previously described). An in vivo assay in which mice were immunized with peptide(s) followed by infectious dengue virus and an in vitro assay of proliferation in response to peptides of T lymphocytes isolated from virus immunized mice were conducted. Peptides 39 (amino acids 137 to 151) and 41 (158-172) elicited high titer peptide and dengue 4 virus binding antibodies in Balb/c mice, as measured by ELISA. Peptides B (30-55), 48 (279-292), 71 (199-212), and 53 (368-382) elicited IgG class peptide-binding antibodies only. Peptide 54 (381-395) appeared to contain a T cell epitope only. In CBA/N or Balb/k mice, peptide 67 (17-30) elicited a response comparable to that of peptides 39 and 41 in Balb/c mice. Peptides 48, 53, and 72 (233-246) elicited high titer IgG peptide antibodies only. The results of the in vitro T cell proliferation assay, using lymphocytes isolated from virus-immunized Balb/c mice, confirmed that peptides 41, 48, 71, and B contain T helper cell epitopes. Surprisingly, peptides 67 and 72 which were positive in the in vivo assay in CBA/N (H-2k) mice, were also recognized in vitro by lymphocytes from virus-immunized Balb/c (H-2d) mice. Lymphocytes from CBA/N mice immunized with virus appeared to recognize only peptide 48 and peptide 30 in vitro. Peptide 30 (1-15) represents a segment in the dengue type 4 E amino-terminal to that of peptide 67 (17-30), which gave the strongest immune response in the in vivo assay. Lymphocytes from peptide 67-immunized CBA/N mice did proliferate when exposed in vitro to peptide 67 and peptide 31 (27-40), which overlaps the carboxy-terminus of peptide 67. These results suggested that peptide 67 (and probably peptides 53 and 72) contains a potential T helper cell epitope at its carboxy-terminus recognizable by H-2k mice but that this sequence is not presented as a normal consequence of processing of E in virus-immunized mice. These results obtained in CBA/N mice await repetition in Balb/k mice (the H-2k strain most congenic with Balb/c). Additional data show that priming with a given peptide in the in vivo assay could result in an anamnestic response to linear epitopes not included on the priming peptide, shortly after the mice were given infectious dengue 4 virus intraperitoneally.